Forming porous scaffold from cellulose derivatives

ABSTRACT

Scaffold comprises a polymer defining macropores and comprising hydroxypropylcellulose partially substituted by a substituent comprising a self-crosslinkable group, which is crosslinked through the self-crosslinkable group. The macropores have an average pore size larger than 50 microns and are at least partially interconnected. In one method, bicontinuous emulsion comprising a continuous aqueous phase and a continuous polymer phase is formed. The polymer phase comprises hydroxypropylcellulose partially substituted by a substituent comprising a self-crosslinkable group, and is crosslinked through the self-crosslinkable group to form a polymer defining at least partially interconnected pores. In another method, phase separation is induced in a solution comprising a polymer precursor and water to form a bicontinuous emulsion comprising a continuous polymer phase and a continuous aqueous phase. The polymer precursor comprises a self-crosslinkable group and is crosslinked through the self-crosslinkable group in the emulsion to form a polymer defining at least partially interconnected macropores.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 12/824,105, filed Jun. 25,2010, which is a continuation-in-part of application Ser. No.12/809,534, filed Jun. 18, 2010, which was the National Stage ofInternational Application No. PCT/SG2008/000491, filed Dec. 18, 2008,which claims the benefit of U.S. Provisional Application No. 61/006,090,filed Dec. 18, 2007, the entire contents of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to porous scaffolds and methods of formingsuch scaffolds.

BACKGROUND OF THE INVENTION

There are many different techniques for forming macroporous scaffolds,such as salt leaching, gas foaming, emulsion freeze drying, fibrousfabric processing, and 3D (three dimensional) printing. Macroporousscaffolds are useful for supporting cells as the large pores canfacilitate migration and growth of the cells inside the pores and allowsufficient nutrient access and removal of metabolites. It has beenreported that when hydroxypropylcellulose (HPC) is crosslinked atdifferent temperatures, both non-porous and microporous HPC hydrogelscan be formed. The average pore sizes in reported microporous HPChydrogels are less than 10 microns. Generally, micropores refer to poresthat have an average pore size in the range of 2 to 50 microns, andmacropores refer to pores that have an average pore size of larger than50 microns.

SUMMARY OF THE INVENTION

It has been recognized that in some applications it is desirable to formmacroporous scaffolds from cellulose derivatives such as HPCderivatives. It has also been surprisingly discovered that macroporousscaffolds can be formed from hydroxypropylcellulose partiallysubstituted by a substituent, where the substituent comprises one ormore self-crosslinkable groups. For example, the substituent can beallyl isocyanate. The macroporous scaffold formed from such partiallysubstituted hydroxypropylcellulose can also have a relatively highinterconnected porosity, such as about 50% or higher. The partiallysubstituted hydroxypropylcellulose may be replaced by anotherthermo-sensitive polymer precursor, such as methylcellulose partiallysubstituted by a substituent that comprises a self-linkable group, or apH-sensitive polymer precursor partially substituted by a substituentthat comprises a self-linkable group.

Therefore, in accordance with an aspect of the present invention, thereis provided a scaffold comprising a polymer defining macropores andcomprising hydroxypropylcellulose partially substituted by asubstituent, the substituent comprising a self-crosslinkable group, thepartially substituted hydroxypropylcellulose being crosslinked throughthe self-crosslinkable group, the macropores having an average pore sizeof larger than 50 microns and being at least partially interconnected.The polymer may have an interconnected porosity of about 50% or higher.The polymer may have a total porosity of about 80% or higher. Themacropores may have a pore size distribution peaking at above 50microns, such as at about 90 or about 100 microns. The polymer may havean equilibrium water content of about 85%. The polymer may have aYoung's modulus of about 10 to about 20 kPa in a hydrated state. Theself-crosslinkable group may comprise an unsaturated doublecarbon-carbon bond. The substituent may comprise allyl isocyanate,methacrylic acid, acrylic acid, or glycidyl methacrylate. The partiallysubstituted hydroxypropylcellulose may have a degree of substitution ofless than about 2.5, such as about 2.1. The polymer may be a gel, suchas when in a hydrated state.

In accordance with another aspect of the present invention, there isprovided a method of forming a scaffold, comprising forming abicontinuous emulsion comprising a continuous aqueous phase and acontinuous polymer phase, the polymer phase comprisinghydroxypropylcellulose partially substituted by a substituent, thesubstituent comprising a self-crosslinkable group; crosslinking thepartially substituted hydroxypropylcellulose through theself-crosslinkable group to form a polymer defining at least partiallyinterconnected pores. The substituent may comprise allyl isocyanate,methacrylic acid, acrylic acid, or glycidyl methacrylate. The pores maycomprise macropores. The crosslinking may comprise irradiating theemulsion with γ-ray. The crosslinking may comprise crosslinking at leastabout 90 wt % of the partially substituted hydroxypropylcellulose in theemulsion. Water may be removed from the pores by freeze-drying thepolymer. After the freeze-drying, the polymer may have an interconnectedporosity of about 50% or higher, and the pores may have an average poresize of larger than 50 microns. The emulsion may comprise about 80 toabout 90 wt % of the aqueous phase and about 10 to about 20 wt % of thepolymer phase. The partially substituted hydroxypropylcellulose may havea degree of substitution of about 2.5 or less, such as about 2.1. Thepolymer may be a gel, such as when in a hydrated state. The emulsion maybe formed by subjecting a solution comprising water and the partiallysubstituted hydroxypropylcellulose to heat treatment. The heat treatmentmay comprise heat treatment at a temperature of about 313 K for about 5minutes.

In accordance with a further aspect of the present invention, there isprovided a method of forming a scaffold, comprising inducing phaseseparation in a solution comprising a polymer precursor and water, toform a bicontinuous emulsion comprising a continuous polymer phase and acontinuous aqueous phase, the polymer precursor comprising aself-crosslinkable group; crosslinking the polymer precursor through theself-crosslinkable group in the emulsion to form a polymer defining atleast partially interconnected macropores. The polymer precursor may bea cellulose derivative. The cellulose derivative may be amethylcellulose derivative, or a hydroxypropylcellulose derivative, suchas hydroxypropylcellulose partially substituted by allyl isocyanate. Thecellulose derivative may be partially substituted by a substituent thatcomprises a self-linkable group. The self-crosslinkable group maycomprise an unsaturated double carbon-carbon bond. The substituent maycomprise allyl isocyanate, methacrylic acid, acrylic acid, or glycidylmethacrylate. The polymer precursor may be thermo-sensitive, and theinducing phase separation may comprise heating the solution. The polymerprecursor may be pH-sensitive, and the inducing phase separation maycomprise changing pH of the solution. The crosslinking may compriseirradiating the emulsion with γ-ray. The polymer may be a gel, such aswhen in a hydrated state.

There is also disclosed a scaffold which comprises a crosslinked polymerthat defines macropores. The macropores are at least partiallyinterconnected and have an average pore size of larger than 50 microns.The interconnected porosity may be about 50% or higher. The polymer isformed from a polymer precursor that is responsive to a phase separationstimuli to undergo phase separation in an aqueous solution. The stimulimay be heat or pH change in the solution. The polymer precursor alsocomprises a self-crosslinkable group so that the polymer molecules arecrosslinked through the self-crosslinkable group. The crosslinkablegroup may be selected so that it will crosslink with each other whenirradiated with γ-ray. The polymer precursor may be a cellulosederivative. The cellulose derivative may be a methylcellulosederivative, or a hydroxypropylcellulose derivative, such ashydroxypropylcellulose partially substituted by allyl isocyanate. Thecellulose derivative may be partially substituted by a substituent thatcomprises a self-linkable group. The self-crosslinkable group maycomprise an unsaturated double carbon-carbon bond. The substituent maycomprise allyl isocyanate, methacrylic acid, acrylic acid, or glycidylmethacrylate. The polymer may have a total porosity of about 80% orhigher. The macropores may have a pore size distribution peaking atabove 50 microns, such as at about 90 or about 100 microns. The polymermay have an equilibrium water content of about 85%. The polymer may havea Young's modulus of about 10 to about 20 kPa in a hydrated state. Thepolymer may have a degree of substitution of less than about 2.5, suchas about 2.1. The polymer may be a gel, such as when in a hydratedstate.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention,

FIG. 1 is a data graph showing the dependence of UV absorbance onsolution temperature for different sample solutions;

FIG. 2 is a scanning electron microscopy (SEM) image of a cross-sectionof a sample scaffold;

FIGS. 3 and 4 are SEM images of portions of the sample scaffold shown inFIG. 2, with increased magnification;

FIG. 5 is a line graph showing the pore size distribution in two samplescaffolds;

FIG. 6 is a confocal micrograph of a cross-section of a sample scaffold;

FIG. 7 is a confocal micrograph of a cross-section of a comparisonscaffold;

FIG. 8 is a confocal micrograph of the sample scaffold of FIG. 6 butstained with a different marker;

FIG. 9 is a confocal micrograph of the sample scaffold of FIG. 8 butwith higher magnification;

FIG. 10 is a data graph showing the dependence of cell numbers in asample scaffold on culture time;

FIGS. 11 and 12 are confocal micrographs of cells cultured in a samplescaffold stained with different markers;

FIG. 13 is a superposition of FIGS. 11 and 12;

FIGS. 14, 15, 16 and 17 are SEM images of cells cultured in a samplescaffold taken at different times with different magnification,respectively;

FIG. 18 is a bar graph showing measured urea synthesis data;

FIGS. 19 and 20 are bar graphs showing measured comparison data;

FIGS. 21, 22, 23 and 24 are SEM images of cells cultured in a samplescaffold;

FIG. 25 is a confocal micrograph (transmitted image) of cells culturedin a sample scaffold;

FIG. 26 is a confocal micrograph of the cells cultured in the samplescaffold of FIG. 25;

FIG. 27 is an SEM image of the cells cultured in the sample scaffold ofFIG. 25 at an earlier time;

FIG. 28 is a confocal micrograph (transmitted image) of the cells ofFIG. 25 at a later time;

FIG. 29 is a confocal micrograph of the cells of FIG. 28;

FIGS. 30, 31, 32 and 33 are confocal micrographs of different cellscultured in different sample scaffolds;

FIG. 34 is a schematic diagram for a synthesis route of a samplescaffold material,

FIG. 35 is a schematic diagram for a process of forming a samplescaffold from the material of FIG. 34;

FIG. 36 is a datagraph showing nuclear magnetic resonance (NMR) spectraof different samples;

FIGS. 37, 38, 39, and 40 are micrographs of a sample scaffold seededwith cells;

FIGS. 37, 38, 39, and 40 are micrographs of a sample scaffold seededwith cells;

FIGS. 41 and 42 are line graphs showing x-ray photoelectron spectra ofdifferent materials;

FIG. 43 shows images of cells cultured on different supportingmaterials;

FIGS. 44, 45, 46, and 47 are SEM images of cells on a pore surface of asample scaffold, exemplary of an embodiment of the present invention;and

FIGS. 48, 49, 50, 51, and 52 are images of stained cells in a samplescaffold, exemplary on an embodiment of the present invention.

DETAILED DESCRIPTION

An exemplary embodiment of the present invention relates to a scaffoldthat is formed of crosslinked hydroxypropylcellulose partiallysubstituted by allyl isocyanate (H-A). A scaffold is any porous materialor structure that can be used to provide a supporting framework tosupport something else, such as cells or tissues. The pores of the H-Apolymerl are macropores of an average pore size of larger than 50microns. The H-A polymer when hydrated may form a gel.

In one embodiment, the H-A polymer has an interconnected porosity ofabout 50% or higher, and may have a total porosity of about 80% orhigher. The interconnected porosity refers to the extent of connectionbetween adjacent pores, and can be determined by mercury intrusionporosimetry.

The macropores may have a pore size distribution peaking at above 50microns, such as at about 90 or about 100 microns. The peak may bedetermined from the pore size distribution curves obtained by mercuryintrusion porosimetry.

The pore sizes and their distribution in a scaffold may be determinedusing a porosimeter, such as a PASCAL 140 mercury porosimeter, availablefrom Thermo Finnigan™.

The pore sizes may be measured when the H-A polymer is in a hydratedstate or dehydrated state, depending on the technique used.

The H-A polymer may have an equilibrium water content (EWC) of about85%, and a Young's modulus of about 10 to about 20 kPa in a hydratedstate. The equilibrium water content (EWC) is calculated asEWC=100%×(W_(h)−W_(d))/W_(h), where W_(d) is the dry weight of the geland W_(h) is the fully hydrated weight of the H-A polymer.

The H-A molecules may have a degree of substitution (DS) of about 2.5 orless, such as about 2.1. The DS may be adjusted by varying the molarratio of —OH in HPC to —NCO in allyl isocyanate. The DS may bedetermined using nuclear magnetic resonance (NMR) spectroscopy, as canbe understood by persons skilled in the art.

Another exemplary embodiment of the present invention relates a processfor preparing a scaffold, such as the scaffold described above. In thisprocess, a bicontinuous emulsion is prepared, which contains acontinuous aqueous phase and a continuous polymer phase. The polymerphase contains H-A molecules as a polymer precursor. The DS of H-A maybe about 2.5 or less, such as about 2.1. The H-A molecules arecrosslinked to form a polymer with at least partially interconnectedmacropores. The emulsion may be irradiated with γ-ray to inducecrosslinking between the H-A molecules. The emulsion may be selected andirradiated such that at least about 90 wt % of the H-A molecules(polymer precursor) are crosslinked (i.e. the degree of crosslink is 90wt %). The H-A polymer will initially form a gel containing water or anaqueous phase in the macropores after crosslinking, which may be removedsuch as by freeze-drying the gel. The contents of the emulsion may beadjusted so that, after freeze-drying, the resulting polymer has aninterconnected porosity of about 50% or higher, and the macropores havean average pore size of larger than 50 microns. The pore sizedistribution may peak at above about 50 microns, such as at about 90 orabout 100 microns. For example, the emulsion may include about 80 toabout 90 wt % of the aqueous phase and about 10 to about 20 wt % of thepolymer phase (or H-A)

Several factors may affect the values of total porosity, interconnectedporosity, and pore size distribution. These factors include polymerprecursor concentration, the DS, crosslinking time, and theconcentration of any crosslinker. These factors may be adjusted tocontrol the porosity of the resulting scaffold. For example, increasingpolymer precursor concentration or crosslinking time may reduce the poresizes of the scaffolds.

In one embodiment, the bi-phase emulsion may be formed by subjecting anaqueous solution of H-A to heat treatment at a temperature of about 313K for about 5 minutes to effect phase separation in the solution.

Conveniently, the scaffolds prepared as described herein can havecertain benefits and advantages and can be used in various applicationssuch as various medical or biological applications.

In some conventional techniques, macroporous hydrogels are prepared incomplex procedures and their pores are not interconnected. The pores inthese conventional hydrogels also lack depth distribution and tend tostay close to the surface. In contrast, embodiments of the presentinvention can provide 3D scaffolds where the interconnected macroporesare distributed substantially uniformly in the entire 3D material. Thescaffold disclosed herein can be prepared using relatively simplechemistry in a relatively simple fabrication procedure. In someembodiments, the process can be performed in a simple one step chemistryunder mild temperature conditions, without using any organic solvent oradditional agent (such as surfactant or crosslinking agent).

As can be appreciated, cellulose (poly(1,4′-anhydro-β-D-glucopyranose))is a major ECM component of plant cells. It is naturally abundant,biocompatible, biodegradable, biorenewable and easily derivatizable.Cellulose-based materials can therefore be conveniently used inpharmaceutical and biomedical applications. The main raw materials usedin embodiments of the present invention, such as HPC, can thus beobtained at a relatively low cost.

Hydroxypropylcellulose (HPC) is a derivative of cellulose and is readilyavailable from commercial sources. It has been approved by the UnitedState Food and Drug Administration (FDA) for use in drug deliveryapplications. HPC and H-A have high solubility in both water and a widerange of organic solvents. They are relatively easy to functionalize tomeet various specific requirements in particular applications. Anaqueous solution of HPC or H-A can undergo a unique low criticalsolution temperature (LCST) phase transition from an isotropic aqueousphase to a metastable bi-phase emulsion. This phase separation allowsthe formation of macroporous polymers and gels.

While microporous hydrogels and nanostructured hydrogels can be formedusing other HPC derivatives, it has been recognized that a macroporoushydrogel would have certain benefits over hydrogels with smaller poresand it has been surprisingly discovered that a bi-phase emulsioncontaining a H-A phase can be conveniently utilized to form macroporoushydrogels.

HPC may be partially substituted with another suitable substituent,instead of ally isocyanate. For example, methacrylic acid, acrylic acid,glycidyl methacrylate, or the like may be a suitable substituent in someapplications.

The inherent thermo-sensitive phase behavior of the H-A polymerprecursor makes it convenient to form bicontinuous polymer-rich andwater-rich phases in the solution by subjecting the solution to heattreatment. Conveniently, it is not necessary to add any chemical solventor agent (e.g. surfactant) to form the bicontinuous emulsion.

The liquid biphasic structure can be stabilized (solidified) bycrosslinking, which in turn may be efficiently effected with γ-rayirradiation. While crosslinking of H-A may be effected using othertechniques, γ-Ray irradiation may be advantageous in some applications.For instance, γ-ray can penetrate deeper into a target material thansome other types of curing light. Thus, crosslinking in the solution canbe activated uniformly at different depths with γ-ray. γ-ray irradiationdoes not give rise to many undesired side chemical effects and do notrequire any chemical initiator. Thus, it is a chemically “clean”technique.

It has been found that H-A with a degree of substitution of about 2.5 orless, such as about 2.1, may be advantageous in some applications. Forexample, H-A polymer precursors with a higher DS tend to have a lowersolubility in water but crosslinked H-A polymer with a lower DS may haveinferior mechanical properties as compared to H-A polymer with a higherDS. Thus, a balance between different desirable properties may beachieved with a DS of about 2.1 in some applications.

The mechanical properties such as mechanical strength of the H-Ascaffolds may be controlled by varying the degree of crosslinkingtherein. The degree of crosslinking may be controlled by varying H-Aconcentration in the precursor solution and the crosslinking time. Theability to control mechanical properties of the H-A scaffolds can beadvantageous in some applications. For example, it may be desirable tobe able to adjust the mechanical properties to provide suitablemechanical stability and structural integrity for supporting cells.

Therefore, the H-A polymer described herein can form three-dimensional(3D) scaffolds for use as soft tissues. The scaffolds can be made tohave various desirable physiochemical, mechanical and bio-interfacialproperties. For example, the scaffold may have properties that aresuitable for promoting cell adhesion. The scaffolds can be maderelatively soft and are inherently hydrophilic. The interconnectedmacropores can facilitate cell growth into the scaffold with celldensities similar to biological tissues. Due to the 3D interconnectedmacropores, the scaffolds not only can have relatively high equilibriumwater content (EWC), be biocompatible, and sustain gradual release ofbioactive molecules, but can also support cell growth, migration, andultimately, tissue formation. The scaffolds also allow readyincorporation of extracellular matrix (ECM) cues to regulate cell andtissue functions. In embodiments of the present invention, the choicesof material and the fabrication procedure can be flexible so that it ispossible to introduce 3D ECM cues for regulation of cellular functions.

As a water soluble precursor can be used for preparing the scaffold, itmay be possible to adjust the degradation profile of the scaffold bymodifying the side chain chemistry of the polymer.

Embodiments of the present invention can also provide 3D scaffolds thathave integrated interconnected macroporosity, nanofeatures (surfacestructures having a nanometer scale dimension, see Examples below), highwater content and mechanical integrity suitable for soft tissueengineering. Such scaffolds can exhibit controllable, moderateelasticity, and hydrophilicity, which can be advantageous in providingmechanical stability and structural integrity to cells and tissues. Thescaffolds can also have nano-features in the pores, which may play arole in the regulation of cell behavior. The nanofeatures may also beuseful for loading of bioactive molecules such as adhesion proteins andgrowth factors etc to control cell behavior and functions.

With interconnected macroporosity and high equilibrium water content,biocompatibility and mechanical integrity, 3D H-A scaffolds as describedherein may be suitable for a wide range of applications in soft tissueengineering and regenerative medicine.

Some embodiments of the present invention may also be suitable forapplication in in vitro substrates for cell culture and studies ofcellular behavior in a 3D environment. 3D cell culture environments moreclosely mimic in vivo microenvironments than 2D culture environments. 3Dscaffolds can therefore serve as ECM analogues to provide biomimicry 3Dsubstrates for cell attachment, growth, migration, function anddifferentiation, or the like.

Some embodiments of the present invention may also be suitable for usein in vitro 3D tissue models that are based upon human cells forpathological study and drug testing.

Some embodiments of the present invention can be used for in vitrocultivation of cells for the creation of external support organs. Thehighly interconnected macroporous architecture can provide temporarysupport to the cultivation of a sufficient cell mass with adequatecell-cell contact. Preliminary results have shown that the primary rathepatocytes cultured in the scaffolds described herein can maintaintheir liver-specific functions over a period of one week (see Examplesbelow). Other types of cells can also be cultivated in these scaffolds.Examples for relevant applications include inartificial liver assisteddevices (BLAD) to provide essential liver functions for the patientswith acute liver failure.

A soft, hydrophilic, and macroporous scaffold may be suitable for celltransplantation in soft tissue and organ repair or regenerationapplications.

Some embodiments of the present invention can be used for high contentscreening for drug discovery and screening. A cellulosic scaffold can beeasily fabricated into microplate configuration to provide uniform 3Dmicroenvironments for various cellular assays.

Some embodiments of the present invention can be used for localized andsustained delivery of biologically or pharmaceutically active compounds.When the scaffolds are hydrophilic and have a high water retentionability, they can be conveniently used to incorporate and providesustained release of bioactive molecules or other substances.

As now can be appreciated, in a different embodiment, HPC may bereplaced with another thermo-sensitive polymer precursor that can form astable bicontinuous emulsion in an aqueous solution in response to heat.For example, methylcellulose may be used to replace HPC. Further, apolymer precursor that can form a stable bicontinuous emulsion in anaqueous solution in response to another type of stimuli, such as achange in pH, may also be used to replace HPC. Thus, a suitablepH-sensitive polymer precursor may be used.

As can also be appreciated, in a different embodiment, allyl isocyanatemay be replaced with another suitable substituent that includes aself-crosslinkable group. Self-crosslinkable groups refer to functionalgroups that can be crosslinked between themselves. For example, asuitable substituent or the crosslinkable group may have an unsaturatedC═C bond for providing a crosslinkable site. A suitable substituentshould also have a functional group for bonding to HPC, so that the HPCcan be partially substituted by the substituent. Suitable replacementfor allyl isocyanate may include methacrylic acid, acrylic acid,glycidyl methacrylate, or the like. The substituent may include amulti-functional monomer.

Depending on the particular substituent used, the degree of substitution(DS) should be adjusted to retain sufficient thermo-sensitivity in thesubstituted HPC to allow convenient phase separation, and to allowsufficient crosslinking of the modified HPC during phase separation.

In view of the disclosure herein, it is possible to provide a scaffoldwhich comprises a crosslinked polymer that defines macropores. Themacropores are at least partially interconnected and have an averagepore size of larger than 50 microns. The interconnected porosity may beabout 50% or higher. The polymer is formed from a polymer precursor thatis responsive to a phase separation stimuli to undergo phase separationin an aqueous solution. The stimuli may be heat or pH change in thesolution. The polymer precursor also comprises a self-crosslinkablegroup so that the polymer are crosslinked through the self-crosslinkablegroup. The crosslinkable group may be selected so that it will crosslinkwith each other when irradiated with γ-ray. The polymer precursor may bea cellulose derivative. The cellulose derivative may be amethylcellulose derivative, or a hydroxypropylcellulose derivative, suchas hydroxypropylcellulose partially substituted by allyl isocyanate. Thecellulose derivative may be partially substituted by a substituent thatcomprises a self-linkable group. The self-crosslinkable group maycomprise an unsaturated double carbon-carbon bond. The substituent maycomprise allyl isocyanate, methacrylic acid, acrylic acid, or glycidylmethacrylate. The polymer may have a total porosity of about 80% orhigher. The macropores may have a pore size distribution peaking atabove 50 microns, such as at about 90 or about 100 microns. The polymermay have an equilibrium water content of about 85%. The polymer may havea Young's modulus of about 10 to about 20 kPa in a hydrated state. Thepolymer may have a degree of substitution of less than about 2.5, suchas about 2.1. The polymer may be a gel, such as when in a hydratedstate.

In some exemplary embodiments, the polymer of the scaffold may includeone or more side chains which include, or are attached to, one or moredifferent functional groups. For example, in one embodiment, the polymermay include hydroxypropylcellulose allyl galactose, where galactoseligands may be present in the side chains. A suitable galactose isβ-galactose. Conveniently, a scaffold formed of hydroxypropylcelluloseallyl galactose can promote formation of cell spheroids when thescaffold is seeded with certain cells, such as primary rat hepatocytecells, as primary rat hepatocyte is one of the cell types that areresponsive to galactose.

Three dimensional (3D) spheroidal formation may be advantageous in someapplications. For example, in liver lobes, there are many stacked layersand some cells will be in the form of spheroids (in vivo) at some pointin time. With spheroids, hepatocyte can have more cell-to-cell contact,which may be useful for polarity maintenance and formation of bilecanaliculi junction (as excretory function). It has been shown that thehepatocytes in spheroids form will have cortical actin (less stressfibers formed), similar to actin in liver lobe.

Galactose can also be a useful ligand for interaction with hepatocytecell membrane (asialo glyco receptor), which can be utilized to improvethe maintenance of hepatocyte differentiated functions.

Conjugating galactose, such as β-galactose, to the scaffold polymer maybe useful for in vitro hepatotoxicity drug testing. To this end, thepore sizes in the scaffold may be selected to be below about 200microns, such as from about 100 to about 150 microns.

A scaffold according to an exemplary embodiment of the present inventioncan be used for testing hydrophobic drugs. In this case, the scaffoldmaterial may include electrically charged molecules or groups conjugatedto the side chains of the scaffold, to reduce adsorption of hydrophobicsubstances onto the scaffold. When the surface material of a scaffoldused to culture the cells is electrically neutral, a significant amountof hydrophobic drug (or protein) can be absorbed on the scaffoldsurface. Thus, to reduce surface absorption, a biocompatible cationicpolymeric group may be conjugated to the side chains of partiallysubstituted hydroxypropylcellulose. The presence of such charged groupscan increase the polarity at the surface, which tends to repel morehydrophobic substances, and thus reduce hydrophobic drug absorption onthe surface. The charged groups may be conjugated onto the side chainsbefore the scaffold is formed or shaped. Potentially useful chargedgroups include polylysine, polyethylene imine, and polypropyleneiminehexadecaamine. Other charged groups or polymers that are known to bebiocompatible with cell culture or gene delivery vector may also be usedas the charged groups, depending on the application.

A biocompatible cationic polymeric group may be attached, such asconjugated to a side chain of the scaffold polymer. Exemplarybiocompatible cationic polymeric groups include polylysine, polyethyleneimine, or polypropyleneimine hexadecaamine. A biocompatible group isnon-toxic to the cells that may come into contact with the scaffold. Forexample, the biocompatible cationic polymeric group may be attached to agalactose ligand in hydroxypropylcellulose allyl galactose.

The side chains of a scaffold polymer may also include other functionalgroups, examples of which include cell attachment ligands such asarginine-glycine-aspartic acid (RGD); ECM for livers such as collagen,laminin, or fibronectin; and cell growth factors such as hepatocytegrowth factors (HGF) conjugated with a spacer. These functional groupsmay be attached or conjugated to the side chains of the polymer or thepartially substituted hydroxypropylcellulose. One or more of thesegroups may also be attached to a surface of the scaffold after itsformation. ECM materials such as collagen Type I, or growth factors, maybe attached to the scaffold surface to adjust, or promote, celldifferentiation.

While some variation and modification of the exemplary embodiments andtheir applications are discussed herein, they are for illustrationpurposes and are not exhaustive. Other applications of the embodimentsof the present invention are also possible.

EXAMPLES

All raw chemical materials used in these examples were obtained fromSigma-Aldrich Pte Ltd.™, Singapore, unless otherwise specified.

Example I Synthesis of Allyl Carbamate of HPC (H-A)

HPC (M_(n)≈10,000; degree of etherification was about 3.4, as determinedby ¹H NMR) was dehydrated by azeotropic distillation in toluene.

The dehydrated HPC (2.0 g, 6.0 mmol [OH]) was dissolved in chloroform(100 ml), to which a solution of allyl isocyanate (1.83 ml, 3.5 molarequivalents) in chloroform (10 ml) was added dropwise. After one drop ofdibutyltin dilaurate was added as a catalyst. the reaction mixture wasstirred at room temperature for about 48 hours, then concentrated usinga rotatory evaporator and precipitated into diethyl ether.

The reaction product was collected by vacuum filtration, and purified byre-dissolution in chloroform and precipitation into diethyl ether. Theresidual impurities were removed by Soxhlet extraction from diethylether.

The product contained, according to ¹H NMR analysis (CDCl₃, δ ppm):0.5-1.5 (—CH₃), 5.7-6.1 (—CH═CH₂), 2.5-5.3 (all other protons).

The degree of substitution was 2.1, as calculated by ¹H NMR recorded inCDCl₃.

The temperature-mediated phase behavior of the sample H-A solutions at10 wt % and 20 wt % (weight percentage of H-A in the solution) wasinvestigated on a UV/VIS/NIR spectrophotometer (Jasco™, V-570, Japan),by measuring the optical densities at 480 nm as a function oftemperature. The temperatures of sample holders were controlled using aJasco PSC-498 temperature controller. The samples were allowed to reachequilibrium at each temperature for 10 min before the readings weretaken. Representative measurement results are shown in FIG. 1. Theoccurrence of phase separation was indicated by reduction in opticaldensity. The temperature at which the reduced optical density reached aplateau was selected as the operating temperature for inducing phaseseparation of H-A solutions and subsequent crosslinking.

FIG. 1 shows representative measured data indicating thetemperature-dependences of normalized (normalized to the absorptionintensity at 298 K) UV absorption of sample solutions containingdifferent concentrations of HPC (diamonds: 10 wt %—solid, 20 wt%—hollow) and H-A (triangles: 10 wt %—solid, 20 wt %—hollow). The phasetransition is manifested by a precipitous decrease in the transmittanceupon increasing temperature. The temperature at which the sampletransmittance decreased by 50% is denoted herein as LCST. Compared toHPC, the phase separation in H-A solutions of both 10 wt % and 20 wt %took place at a lower temperature range, with LCST being about 307 K,resulting from hydrophobic substitution with allyl groups.

Like the HPC samples, the H-A samples became white and opaque when thetemperature was increased, but without any noticeable sedimentation overthe temperature range studied, indicating the formation of stablecolloidal systems in the H-A solutions.

Stable colloidal formation can be advantageous for subsequent generationof 3D open porous structures.

Based on the experimental data, 313 K was selected as a suitabletemperature for induction of phase separation and crosslinking of H-Asolutions.

Example II Preparation of 3D H-A Scaffolds

Sample solutions of H-A and water were prepared. The solutions containedabout 10 or about 20 wt % of H-A respectively. The water was degassedand deionised before mixed with H-A.

In a representative procedure, a glass vials (10 mm in diameter×50 mmheight) containing a sample H-A solution was placed in a water bath at313 K for 5 min to induce phase separation in the solution to form abicontinuous emulsion.

The emulsion was transferred, in a beaker containing water at 313 K, toa gamma irradiator (Gammacell 220, MDS Nordion™, Canada), and subjectedto γ-ray irradiation at a dose rate of 10 kGy h⁻¹ for 30 min, tocrosslink the polymeric phase in the emulsion to form a gel.

The crosslinked gel was freeze-dried, washed with deionised water forone week with daily water change, and lyophilized for storage.

The sample polymer (scaffold) formed with 10 wt % H-A is referred to asSample I, and the sample polymer (scaffold) formed with 20 wt % H-A isreferred to as Sample II.

Example III Comparison Samples

For comparison purposes, H-A solutions as prepared in Example II werealso crosslinked at temperatures from about 273 to about 277 K, withoutphase separation, by irradiation as described in Example II. Theresulting products were homogenous gels and were subjected to similarpost-irradiation treatment as described in Example II.

These gels were referred to as Sample IC (for 10 wt % H-A) and SampleIIC (for 20 wt % H-A).

Example IV Characterization of Sample Scaffolds

The degree of crosslink in the samples was expressed as the weightpercentage of the crosslinked fraction of the scaffold.

Residual soluble polymers were removed by Soxhlet extraction withacetone for 4 h.

The lyophilized scaffolds were allowed to swell in water at roomtemperature for 48 h. The samples were weighed before and after thishydration process. The EWC of the sample scaffolds were determined basedon their dry and hydrated weights, according to the equation discussedabove.

The Mechanical properties of hydrated scaffolds were evaluated bycompression tests on an Instron™ Micro-Tester 5848 (Instron Co., Canton,Mass., U.S.A.), at a speed of 0.5 mm/min and a temperature of 25±2° C.The compression moduli were calculated from the slopes of the initiallinear portion of the stress-strain curves.

The pore size distribution in the sample scaffolds was determined usinga PASCAL 140 mercury porosimeter (Thermo Finnigan. Italy, S.p.A.) withS-CD6 dilatometer.

Cross-sectional surfaces of sample lyophilized H-A scaffolds weresputter coated with gold and examined on a field-emission scanningelectron microscope (FESEM) (JEOL™, JSM-7400M, Japan) at an acceleratingvoltage of 20 kV. The morphology of the hydrated H-A scaffolds wasinvestigated by laser confocal fluorescence microscopy. The freeze-driedscaffolds were stained overnight with fluorescein isothiocyanateconjugated dextran (FITC-dextran) (1.0 mg ml⁻¹) or propdium iodide (PI)(50 μg ml⁻¹) in the phosphate-buffered saline (PBS), respectively. Theywere then washed with PBS for 5 times and examined using a laserconfocal fluorescence microscope (Olympus™ Fluoview 300, Japan). Opticalsectioning with a Z resolution of 2 μM was used to obtain a 3D imagestack of scaffolds.

Some representative results of the above tests are summarized in Table Iand shown in the figures.

TABLE I Properties of Sample H-A Scaffolds Sample I Sample II Porosity(%) 83 92 Interconnected porosity (%) 52 56 Peak pore size (micron) 10388 EWC (wt %) 91 89 Young's modulus in hydrated state (kPa) 9.5 20.5Degree of crosslink (wt %) 90 92

In Table I, peak pore size refers to the location of the highest peak inthe pore distribution, and is the pore size that occurs most frequentlyin a porous material.

There was no significant observed change in sample dimension due todehydration or re-hydration. This stability is expected to be due to theextensive (about 90 wt %) crosslinking in the H-A polymer.

The presence of interconnected macroporous structures in Samples I andII was observed by mercury intrusion porosimetry and scanning electronmicroscopy (SEM). FIG. 2 is a representative SEM image of Sample II at amagnification factor of 90. Further magnified SEM images of a portion ofthe image of FIG. 2 are shown in FIGS. 3 (500 magnification) and 4(15000 magnification). Similar interior morphology of interconnectedmacroporosity were also observed in Sample I. Nano-scale features andstructures on the pore surfaces, such as edges, spikes in the regionsconnecting the macropores, were also visible in the images.

In contrast to some conventional techniques in which only microporeswere formed in HPC scaffolds, the sample scaffolds formed ofhydrophobically modified H-A had interconnected macropores. The H-Asamples also retained the phase behavior characteristics of HPC, asindicated by the formation of stable, opaque colloidal system over thetemperature range studied.

Both samples had a broad and bimodal distribution of pore sizes, asshown in FIG. 5 (Sample I—solid; Sample II—hollow), with 50% to 60%interconnected porosity respectively. As can be seen, for both samples,the pore size distribution had two peaks, a taller and sharper peak at asmaller pore size and a shorter and broader peak at a larger pore size.When the H-A concentration was increased, from about 10 wt % to about 20wt %, the taller peak in the pore size distribution curve was shifted tothe left (smaller pore size) and the higher peak was shifted to theright (larger pore size); and the taller peak became taller and theshorter peak became shorter.

The porous structures of Samples II and IIC were also characterized andcompared in their hydrated state.

Both Samples II and IIC were stained with FITC-dextran in PBS for 24hours. A highly porous network was observed in hydrated Sample I inlaser confocal microscopy images of the sample. See a representativeimage in FIG. 6. In comparison, FIG. 7 shows a similar image for SampleIIC, which revealed a nonporous, homogenous structure. This resultindicates that phase separation facilitated the formation ofinterconnected macropores.

The highly macroporous architecture in hydrated Sample II was alsoconfirmed in another staining study using propidium iodide (PI).Representative images of PI-stained Sample II are shown in FIGS. 8 and9. The sizes of water-filled pores varied broadly from tens to hundredsmicrons and the thickness of water-swollen struts ranged from about 5 toabout 20 microns.

Example V Cell Culture and Seeding in Sample Scaffolds

For cell culture, Samples I and II were cut into discs of about 10 mm indiameter and about 2 mm in thickness.

NIH 3T3, HepG-2 and MCF-7 cells (ATCC, USA) were cultured according tothe standard procedure in high glucose Dulbecco's modified Eagle'smedium (DMEM) (Invitrogen™, Singapore) supplemented with 10% fetalbovine serum, 100 unit/ml penicillin and 100 μg/ml streptomycin in ahumid incubator at 37° C. and 5% CO₂, respectively.

Hepatocytes were harvested from male Wistar rats weighing 250-300 g by atwo-step in situ collagenase perfusion method, as described in P. O,Seglen, Methds Cell Biol., 1976, vol. 13, p. 29. Viability of thehepatocytes was ≧90%, as determined by Trypan Blue Exclusion assay.Hepatocytes cultured in the scaffolds were maintained in William's Esupplemented with 1 mg/ml bovine serum albumin (BSA), 10 ng/ml epidermalgrowth factor (EGF), 0.5 μg/ml insulin, 5 nM dexamethasone, 50 ng/mllinoleic acid, 100 unit/ml penicillin and 100 μg/ml streptomycin.

The sample scaffold discs were sterilized in a 12 well plate by gammairradiation at a dose rate of 10 kGy/h for 4 h. Cell seeding wasconducted at a density of 0.5˜1.0×10⁶ cells per scaffold, by adding 20μl of concentrated cell suspension into each scaffold followed by 200 μlof cell medium. Cell-free scaffold was used as control for all thefollowing work. The scaffolds were incubated for 3 h, after which time 1ml of cell medium was added, respectively. The viability of cellscultivated in the scaffolds was assessed by fluorescence live/deadstaining. The scaffolds were incubated for 30 min with 5 μM Calcein AM(Molecular probe, USA) and 25 μg/ml of propdium iodide in Dulbecco'smodified essential medium (DMEM) at 37° C., 5% CO₂. Images of live(green) and dead (red) cells were then acquired by laser confocalfluorescence microscopy. Optical sectioning with a Z-resolution of 2 μmwas used to obtain a 3D image stack of scaffolds.

Proliferation of NIH 3T3 seeded in sample scaffolds were assessed bymonitoring their metabolic activities using almarBlue™ assay. Thecell-seeded and cell-free scaffolds were incubated for 4 hours with 10%(v/v) almarBlue (Biosource) in phenol-red free, supplemented DMEMmedium. 200 μl of the media from each sample was transferred to a96-well plate, and the absorbance at 570 nm and 600 nm were measuredusing a Sunrise™ microplate reader (Tecan, Switzerland). The reductionof almarBlue of each sample was calculated, and converted to cellnumbers based upon a standard curve constructed from 2D cell culturewith known cell numbers.

Sample II was selected for cellular compatibility tests. A detailed cellviability and proliferation test was conducted on the mouse fibroblastNIH 3T3 cultivated in the scaffold, by measuring the metabolic activityusing Alamar Blue assay. The cells were found to be not only viable butalso to proliferate well over prolonged culture. The number of cellsover time in Sample II was plotted in the graph of FIG. 10.

FIG. 11 is a fluorescence microscopy image of NIH 3T3 cells cultured inSample II after 4 weeks of cultivation. The cells were stained withCalcein AM (shown as bright regions in FIG. 11) for live cells. FIG. 12is a similar image but for cells stained with PI (shown as lighter spotsin FIG. 12) for dead cells. FIG. 13 is a superposition of the images ofFIGS. 11 and 12.

FIGS. 14 and 15 show SEM images of NIH 3T3 cultured in Sample II for oneday at different magnifications. FIGS. 16 and 17 show SEM images of NIH3T3 cultured in Sample II for five days at different magnifications. Thescale bars represent 100 microns in FIGS. 14 and 16, and 10 microns inFIGS. 15 and 17.

To investigate the urea synthesis of hepatocytes, the cells cultured inthe sample scaffolds were incubated in a culture medium containing 1.0mM NH₄Cl for 90 min; the medium was analyzed by the Urea Nitrogen Kit(Sigma Diagnostics), as described in S. Ng et al., Biomaterials, 2005,vol. 26, p. 3163. The data was normalized by the number of cells seededin the scaffolds that was quantified using Quan-iT™ PicoGreen dsDNAAssay Kit (Invitrogen, Singapore).

Primary hepatocyte has been a major cell source for liver tissueengineering; it tends to lose its hepatic function rapidly in vitro. Thepresent data showed that primary rat hepatocytes cultured in the samplescaffolds could maintain their urea secretion, a liver-specificfunction, over a period of one week. FIG. 18 shows the measured result.

FIGS. 19 and 20 show albulmin secretion and urea synthesis of theprimary rat hepatocytes cultured in Sample II, in comparison to 2Dcollagen monolayer culture. 2D collagen monolayer has beenconventionally used for hepatocyte culture. The sustained liver-specificfunctions of primary rat hepatocytes cultured in the scaffolds weredifferent in the two tested scaffolds. The albumin secretion and ureaproducts of the hepatocytes cultivated in the 3D Sample II were higherthan those cultured in the 2D collagen monolayer.

FIGS. 21, 22, 23 and 24 show SEM images of primary rat hepatocytescultured in Sample II, taken over a period of 7 days, and themorphologies of these cells in Sample II. The cells appeared round andtended to form cellular aggregates in the scaffold, well supported bycell-cell and cell-matrix interactions.

Fluorescence viability staining was also carried out on human MCF-7breast cancer cells (MCF-7), human hepatoblastoma cells (C3A), showinggood cellular biocompatibility.

FIG. 25 is a transmitted image of C3A cells cultured in sample 3Dscaffolds for two days. The C3A cells formed spheroids in the scaffolds.FIG. 26 is a fluorescence image of live/dead staining of the C3Aspheroids at day 2. FIG. 27 is an SEM image of the C3A spheroids at day1 and FIG. 28 is a transmitted image of the C3A spheroids at day 7. FIG.29 is a fluorescent image of live/dead staining of C3A spheroids at day7.

It is expected that the presence of —OH groups in Sample scaffolds canalso allow facile incorporation of ECM proteins to improve celladhesion. This method is also applicable to other types of ECM proteins.Collagen type I was used to verify this expectation. Under sterilecondition, scaffolds were rinsed with acetone for three times, thentreated with 20 mM 1,1′-carbonyl diimidazole (CDI) overnight at roomtemperature. It is expected this method is also applicable to othertypes of ECM proteins. The scaffolds were washed with acetone for 5times before being immersed into collagen I solution (0.29 mg/ml,pH10.0). The conjugation was conducted on an orbital shaker at 4° C.overnight. The modified scaffolds were then washed with PBS anddeionised water before freeze dry. Cell attachment has beensignificantly improved in the modified scaffolds as demonstrated inFIGS. 31 and 33. in FIG. 30 shows an image of live/dead staining ofhuman foreskin fibroblasts cultured in an unmodified sample scaffold atday 1; FIG. 31 shows an image of live/dead staining of human foreskinfibroblasts cultured in an collagen conjugated sample scaffold at day 1;FIG. 32 shows an image of live/dead staining of human umbilical veinendothelial cells cultured in the unmodified scaffold at day 1; and FIG.33 shows an image of live/dead staining of human umbilical veinendothelial cells cultured in the collagen conjugated sample scaffold atday 1.

Example VI Galactosylated Hydroxypropyl Cellulose Scaffold Synthesis andFabrication

The synthesis procedure of galactosylated hydroxypropyl cellulose (HAGal) scaffold is illustrated in the schematic diagram of FIG. 34. InFIG. 34, compound 1 is hydroxypropyl cellulose parent molecule, compound2 is hydroxypropyl cellulose allyl, compound 3 is intermediate moleculeof hydroxypropyl cellulose modified with imidazole group, and compound 4is hydroxypropyl cellulose allyl galactose.

To obtain hydroxypropyl cellulose allyl basic construct, 4 gr of driedhydroxypropyl cellulose (HPC MW 80,000 Da, Sigma Aldrich) was dissolvedin 100 ml anhydrous chloroform (Sigma Aldrich™). The mixture was stirredfor 1 day at room temperature until completely dissolved. On the nextday, 50 ml anhydrous chloroform was added to the mixture to reduceviscosity. 2.095 ml allyl isocyanate 98% (MW 83.04 Da, ρ=0.946 g/cm³,Sigma Aldrich) dissolved in 2 ml anhydrous chloroform was added dropwiseto the mixture, as a side chain modifier for crosslinking through doublebonds. Dibutyltin dilaurate 95% (2 ml, Sigma Aldrich) was added to themixture as a catalyst. The mixture was stirred for 48 hours at roomtemperature in sealed flask to prevent moisture. Two days later, themixture was concentrated in rotary evaporation at 35° C. andprecipitated in anhydrous diethyl ether. The fibrous end-product wasfurther vacuum dried for half a day to remove ether traces.

Dialysis was performed in tube with molecular cut off 12 kDa-14 kDa for3 days in water to remove impurities. The final product was then freezedried. This product was denoted as HA (hydroxypropyl cellulose allyl).

HA (1 gram) was dissolve in 15 ml anhydrous dimethylformamide. Equimolarof 1,1′-carbonyldiimidazole (CDI, 0.322 g) dissolved in 2 ml anhydrousdimethylformamide was added once the HA had been completely dissolved.Activation of HA hydroxyl groups was performed with CDI for 2 hours atroom temperature.

Equimolar of fully dissolved β-D-galactosamine HCl (0.427 g in 30 mlanhydrous dimethylformamide with addition of 500 μl triethylamine assalt-form releaser) was added and reacted for 48 hours. The mixture wasthen precipitated in excess anhydrous diethyl ether until yellowishslurry was obtained. This slurry was further vacuum dried to removetraces of ether. Dried product was further dissolved in water anddialyzed to remove impurities (dialysis tube with molecular cut off 12kDa-14 kDa) for 3 days. The product was then freeze dried and kept indesiccators until use.

This product was denoted as HA Gal (hydroxypropyl cellulose allylgalactose).

As schematically illustrated in FIG. 35, purified HA Gal product wasdissolved in deonized water at concentration 7.5 wt/vol %. Thecompletely dissolved mixture was poured into small glass tubes (6 mmdiameter) at 3 cm height. Bubble-free solution in glass tubes wereclustered in bigger vessel and put in 50° C. water bath for 5 min toinduce stable colloidal formation. When the solution temperature wasincreased to above the LCST, phase transition occurred. The clear,transparent solution turned into an opaque colloidal solution at above45° C. The colloids formed were crosslinked with gamma irradiation for30 min at 10 kGray/hour. The crosslinked products in the tubes wereextracted by breaking the glass tubes at freezing temperature. Theproducts were then sliced evenly at 1 mm thickness in water withKrundieck tissue slicer. These slices were further freeze dried toremove the water and gamma irradiated to sterilize.

The final scaffold products were able to fit 96 well-plates and wereready for cell plating.

Representative NMR measurement results of HA Gal, HA and HPC samples areshown in FIG. 36. The NMR spectra of HA Gal show visible peaks around 7ppm, which indicates formation of amide bond between galactose andhydroxypropyl cellulose through imidazole group. The NMR spectra of HAshow visible peaks around 4.5-5 ppm, which indicate existence of allylgroup bonds. The bottom NMR spectra in FIG. 35 are for parent moleculehydroxypropyl cellulose (HPC).

Images of a sample HA Gal scaffold seeded with primary rat hepatocytewere taken at different times after initial seeding. Representativeimages taken after different seeding periods are shown in FIG. 37 (day1), FIG. 38 (day 3), FIG. 39 (day 5), and FIG. 40 (day 7). The imagesshow that 3D spheroids were formed as early as 24 hours after initialseeding.

Representative X-ray photonelectron speactra (XPS) of HA and HA Gal areshown in FIGS. 41 (HA) and 42 (HA Gal), respectively. As can be seen,the counts at both 280 eV (Cls) and 550 ev (O) positions are higher forHA Gal than for HA.

FIG. 43 compares images of hepatocyte cells cultured on differentsupporting materials, which were, from the left column to the rightcolumn, collagen monolayer, PET Gal membrane, and sample HA Galscaffold. The images were taken on day 1, 3 or 6 (from top row to bottomrow), respectively. The scale bars shown in FIG. 43 represent 60 micronsin length. As can be seen, the cells cultured in HA Gal scaffold startedto form cell spheroids on day 1. In comparison, visible cell spheroidswere formed in PET Gal membrane on after day 3.

FIGS. 44, 45, 46, and 47 show SEM images of primary rat hepatocytes onthe sample HA gal scaffold. Images taken at different times after cellseeding are shown in FIG. 44 (day 1), FIG. 45 (day 3), and FIG. 46 (day17). FIG. 47 shows a single cell tethered onto the nanoscale surfacestructure of the sample scaffold.

FIGS. 48, 49, 50, 51, and 52 show images of F-actin stained rathepatocyte spheroids formed on the sample HA Gal scaffold.

Other features, benefits and advantages of the embodiments describedherein not expressly mentioned above can be understood from thisdescription and the drawings by those skilled in the art.

Of course, the above described embodiments are intended to beillustrative only and in no way limiting. The described embodiments aresusceptible to many modifications of form, arrangement of parts, detailsand order of operation. The invention, rather, is intended to encompassall such modification within its scope, as defined by the claims.

1. A method of forming a scaffold, comprising: forming a bicontinuousemulsion comprising a continuous aqueous phase and a continuous polymerphase, said polymer phase comprising hydroxypropylcellulose partiallysubstituted by a substituent, said substituent comprising aself-crosslinkable group; crosslinking said partially substitutedhydroxypropylcellulose through said self-crosslinkable group to form apolymer defining at least partially interconnected pores.
 2. The methodof claim 1, wherein said substituent comprises allyl isocyanate,methacrylic acid, acrylic acid, or glycidyl methacrylate.
 3. The methodof claim 1, wherein said pores comprise macropores.
 4. The method ofclaim 1, wherein said crosslinking comprises irradiating said emulsionwith γ-ray.
 5. The method of claim 1, wherein said crosslinkingcomprises crosslinking at least about 90 wt % of said partiallysubstituted hydroxypropylcellulose in said emulsion.
 6. The method ofclaim 1, comprising removing water from said pores by freeze-drying saidpolymer, wherein, after said freeze-drying, said polymer has aninterconnected porosity of about 50% or higher, and said pores have anaverage pore size of larger than 50 microns.
 7. The method of claim 1,wherein said emulsion comprises about 80 to about 90 wt % of saidaqueous phase and about 10 to about 20 wt % of said polymer phase. 8.The method of claim 1, wherein said partially substitutedhydroxypropylcellulose has a degree of substitution of about 2.5 orless.
 9. The method of claim 1, wherein said polymer is a gel.
 10. Themethod of claim 1, wherein said emulsion is formed by subjecting asolution comprising water and said partially substitutedhydroxypropylcellulose to heat treatment at a temperature of about 313 Kfor about 5 minutes.
 11. The method of claim 1, wherein said partiallysubstituted hydroxypropylcellulose comprises hydroxypropylcelluloseallyl galactose, and said method comprises attaching a biocompatiblecationic polymeric group to a side chain of said hydroxypropylcelluloseallyl galactose, wherein said biocompatible cationic polymeric groupcomprises polylysine, polyethylene imine, or polypropyleneiminehexadecaamine.
 12. The method of claim 1, comprising attaching anarginine-glycine-aspartic acid (RGD), collagen, laminin, fibronectin, orcell growth factor to a surface of said scaffold.
 13. A method offorming a scaffold, comprising: inducing phase separation in a solutioncomprising a polymer precursor and water, to form a bicontinuousemulsion comprising a continuous polymer phase and a continuous aqueousphase, said polymer precursor comprising a self-crosslinkable group; andcrosslinking said polymer precursor through said self-crosslinkablegroup in said emulsion to form a polymer defining at least partiallyinterconnected macropores.
 14. The method of claim 13, wherein saidpolymer precursor is a cellulose derivative.
 15. The method of claim 14,wherein said cellulose derivative is a methylcellulose derivative or ahydroxypropylcellulose derivative, or wherein said cellulose derivativeis partially substituted by a substituent that comprises a self-linkablegroup.
 16. The method of claim 15, wherein said hydroxypropylcellulosederivative is hydroxypropylcellulose partially substituted by allylisocyanate, or comprises hydroxypropylcellulose allyl galactose.
 17. Themethod of claim 13, wherein said self-crosslinkable group comprises anunsaturated double carbon-carbon bond.
 18. The method of claim 13,wherein said substituent comprises allyl isocyanate, methacrylic acid,acrylic acid, or glycidyl methacrylate.
 19. The method of claim 13,wherein said polymer precursor is thermo-sensitive, and said inducingphase separation comprises heating said solution; or said polymerprecursor is pH-sensitive, and said inducing phase separation compriseschanging pH of said solution.
 20. The method of claim 13, wherein saidcrosslinking comprises irradiating said emulsion with γ-ray.
 21. Themethod of claim 13, wherein said polymer is a gel.